Leishmania infantum transfected with toxic plasmid induces protection in mice infected with wild type L. infantum or L. amazonensis

Author(s):Show less -

Zorgi, Nahiara Esteves ^[1] ; Arruda, Leonardo V. ^[2] ; Paladine, Izadora ^[1] ; Roque, Guilherme A. S. ^[1] ; Araujo, Thalita F. ^[1] ; Brocchi, Marcelo ^[3] ; Barral, Manoel ^{[4, 2]} ; Sanchiz, Africa ^[5] ; Maria Requena, Jose ^[5] ; Abanades, Daniel R. ^[1] ; Giorgio, Selma ^[1]

Affiliation

^[1] Univ Estadual Campinas, Inst Biol, Dept Anim Biol, Campinas, SP - Brazil

^[2] Fundacao Oswaldo Cruz, Res Ctr Goncalo Moniz, Salvador, BA - Brazil

^[3] Univ Estadual Campinas, Inst Biol, Dept Microbiol & Immunol, Campinas, SP - Brazil

^[4] Univ Fed Bahia, Sch Med, Salvador, BA - Brazil

^[5] Autonomous Univ Madrid, Dept Mol Biol, Ctr Mol Biol Severo Ochoa, Madrid - Spain

• Document type: Journal article
• Source: Molecular Immunology; v. 127, p. 95-106, NOV 2020.
• Web of Science Citations: 0

Abstract

Leishmania infantum infection may cause visceral leishmaniasis (VL), a fatal disease having worldwide distribution, that may be silent or asymptomatic. The latter indicates that immunity is naturally developed in some individuals, and, therefore, a vaccine against VL would be possible. Molecular mechanisms of gene expression are being understood in Leishmania, and this knowledge may be useful for vaccine development. The aim of this study was developing an attenuated strain by regulating the expression of toxic proteins in a stage specific manner. For that purpose, the 3' UTR of an amastin gene, known by its increased expression in the amastigote phase, was selected for direct the expression of exogenous proteins. This construct (pFL-AMA), firstly, was proved effective for the expression of mCherry specifically in the intracellular form of L. infantum, as demonstrated by fluorescence microscopy, flow cytometry and Western blotting. Afterwards, mCherry coding sequence was replaced, in the pFL-AMA plasmid, by either egg avidin or the active form of bovine trypsin. Viability of transfected parasites was evaluated in promastigote axenic cultures and in in vitro infection of macrophages. Both lines of transfected parasites showed a limited capacity to multiply inside macrophages. BALB/c mice were inoculated intraperitoneally (i.p.) with a single dose consisting of 2 x 10(6) L. infantum promastigotes transfected with plasmids bearing the toxic genes. After 10 weeks post-inoculation, no parasites were recovered by limiting dilution in either liver or spleen, but a specific immunological response was detected. The immunization with transfected parasites induced cellular and humoral immune responses with activation of TCD4(+), TCD8(+) and B cells, having a TH1-type response with increased levels of pro-inflammatory cytokines such as IFN-gamma, TNF-alpha and IL-6. In parallel groups of mice, a challenge consisting on 1 x 10(6) virulent parasites of either L. infantum (inoculated i.p.) or L. amazonensis subcutaneously (s.c.) was performed. Vaccinated mice, challenged with L. infantum, showed lower parasite burdens in liver, spleen and bone marrow than infected mice with WT L. infantum (non-vaccinated); similarly, vaccinated mice developed smaller footpad inflammation than control group. These data support this strategy as an efficient immunization system aimed to the development of vaccines against different forms of leishmaniasis. (AU)

• FAPESP's process: 18/23302-6 - Leishmaniasis of medical importance in Brazil: study of cellular systems and clinical isolates of Leishmania infantum and L. braziliensis and evaluation of vaccine candidates against L. infantum
• Grantee: Selma Giorgio
• Support Opportunities: Regular Research Grants
• FAPESP's process: 19/06322-6 - Evaluation of gene products encoded in L. infantum parasites transfected with the plasmids pFL-AMA-AVITOX and pFL-AMA-TRYPTOX after the differentiation process
• Grantee: Nahiara Esteves Zorgi
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• FAPESP's process: 15/23767-0 - Development of biomarkers and Leishmania infantum strains
• Grantee: Selma Giorgio
• Support Opportunities: Regular Research Grants
• FAPESP's process: 16/11539-6 - Study of immunoprotective capacity induced by infection with "suicide" strains of Leishmania infantum in mice
• Grantee: Nahiara Esteves Zorgi
• Support Opportunities: Scholarships in Brazil - Post-Doctoral